Digitalis preparations, namely digoxin and digitoxin are used extensively in the therapy of congestive heart failure and certain disturbances of cardiac rhythm. The dosage of digitalis preparations; however, must be carefully adjusted to the needs of these patients so that optimal therapeutic effect is achieved without the development of toxic side-effects. Certain developments have enhanced the physician's ability to adjust the dosage of digitalis preparations to the needs of each patient, and such developments have included; the introduction into clinical use of highly purified cardiac glycosides derived from digitalis leaf; the recognition of the effects of cardiac glycosides on myocardial contractility, conduction in automaticity; and appreciation of the role of electrolyte imbalance in facilitating the development of digitalis toxicity; and a better understanding of the absorption, metabolic fate; and excretion of digitalis based on studies with radioactively labeled cardiac glycosides.
Preparation of isotopically labeled digitalis derivatives allows development of methods for accurate quantitation of nanogram (1X10.sup.-.sup.9) amounts of .sup.14 C or .sup.3 H labeled digitoxin and digoxin. The use of radiolabeled digitalis preparations made possible for the first time the direct measurement of cardiac glycosides and their metabolites in the blood and tissues of man and experimental animals. Among many important findings were the observations that serum digoxin concentrations in subjects on usual therapeutic doses were of the order of 1 to 2 ng/ml.sup.4 and for digitoxin approximately tenfold higher.
Several methods or techniques are currently used for determining serum or plasma concentrations of cardiac glycosides, and one such technique is the radioimmunoassay technique. In the radioimmunoassay technique, there is competition between unlabeled molecules present in the fluid to be assayed and a radioactively labeled form of the substance, added in vitro, for a limited number of specific antibody binding sites. At equilibrium, the ratio between antibody bound and free labeled substance is a sensitive indicator of the amount of unlabeled substance originally present in the sample. The radioimmunoassay technique has been used in the measurement of serum levels of both digitoxin and digoxin in the range encountered clinically.
The radioimmunoassay method is the simplest method in current use. It employs whole serum, without the need for an extraction step, and involves the addition of three reagents followed by a single centrifugation step. It is the most rapid; a single serum can be analyzed in one hour and several dozen sera can be assayed in a slightly longer period. Because it is the most sensitive, determinations can be performed on as little as 0.1 ml. of serum. Because of its simplicity (particularly in that no extraction step is required), rapidity, and sensitivity, the radioimmunoassay method is the most convenient for routine clinical determinations of serum concentrations of digoxin and digitoxin. Its principal disadvantage is that it employs liquid scintillation counting, with the resultant problems of quenching and chemiluminescence, problems that have been overcome by the use of radioiodinated digitalis derivatives.
The cardiac glycosides are all relatively small molecules with molecular weights in the 500-1000 range and are too small to be immunogenic by themselves. Therefore, in order to obtain antibodies to cardiac glycosides, it is necessary to conjugate these pharmacologic agents as haptens to antigenic protein carriers. For this purpose, periodate oxidized glycosides have been coupled to albumin carriers to form synthetic glycoside-protein conjugates. Alternatively, the 3-O-succinyl derivatives of digitoxigenin and digoxigenin, the cardioactive steroidal aglycone portions of the corresponding glycosides, lacking the 3 digitoxose residues have been conjugated to albumin carriers by carbodiimide and mixed anhydride methods. Rabbits and other animals immunized with such conjugates formed antibodies to the protein carrier and also formed antibodies to the glycoside hapten. Antibodies to digoxin produced in this manner have been shown to have a high affinity and great specificity for this glycoside. For example, antidigoxin antibodies bind digoxin at least twenty times more effectively than they bind digitoxin, although digitoxin differs structurally from digoxin only in the absence of a single hydroxyl group at the C-12 position.
In the radioimmunoassay procedure, the non-radioactive glycoside will compete with the radioactive labeled glycoside for combining sites on antidigitalis antibodies.
When varying quantities of unlabeled digitalis are mixed with a standard amount of radio labeled glycoside, the amount of radioactivity bound by a standard amount of antibody will decrease as increasing amounts of unlabeled glycosides are added. A standard curve can then be constructed from which the concentration of digitalis in a given patient serum can be determined on the basis of the decrease that it causes in the binding of radioactive glycoside by a specific antibody.
In certain clinical situations, it is necessary to obtain digoxin results as quickly as possible. The unavailability of the history of digoxin dosage, and in cases of malabsorption and fluctuating renal function, are typical clinical situations that arise in which rapid results are required. It has been determined that quantitation of the cardiac glycosides in serum or plasma is most conveniently accomplished through radioimmunoassay. This assay method is the most rapid, precise sensitive and specific, and at the same time can readily cope with a large number of patient samples. The use of iodinated digoxin further increases assay sensitivity, shortens counting time, and eliminates the problems with the liquid scintillation counting of beta emitting .sup.3 H (tritiated) glycosides. Thus, the major practical contribution of the use of iodinated cardiac glycosides is a combination of counting reliability and assay speed.
There are commercially prepared radioiodinated derivatives of digoxin and digitoxin for use in radioimmunoassay tests, but these prior art radioiodinated digitalis derivatives have certain disadvantages. The principle disadvantage of these prior art compounds is the steric hindrance involved during the antigen-antibody reaction.